The amplification of complex template nucleic acids plays an important role in many molecular-biological applications. Thus, for some applications, it is necessary to amplify whole genomes (Whole Genome Amplification, WGA), whole transcriptomes (Whole Transciptome Amplification, WTA) or whole bisulfitomes (Whole Bisulfitomes Amplification, WBA), respectively.
Various parameters are essential for the successful amplification of such complex nucleic acids:
Suitable reaction conditions are a necessary requirement. These include, e.g., a suitable buffer with a suitable pH value and a suitable composition of monovalent and bivalent salts. However, they also include at least one suitable polymerase and suitable reaction temperatures for the amplification are essential factors for the success of the amplification.
A decisive parameter is the amount of complex template nucleic acid used. If the amount of complex template nucleic acid is too small, it will not suffice for the whole genome. For example, an amount of 6 pg human genomic DNA corresponds approximately to the haploid human genome. If less than 6 pg are used for one WGA reaction, it is not possible to completely amplify the human genome in the WGA since not all segments are represented. At the same time, an amount of less than 100 pg can also be too small since specific segments from stochastic effects are under- or overrepresented in such an amount.
A third decisive parameter for the amplification of complex template nucleic acids is the quality of the sample or the complex template nucleic acid(s) contained therein, respectively. The term “quality” may include different aspects:
The sample to be amplified containing complex template nucleic acid can have inhibitors which inhibit the polymerase used in a competitive or allosteric way or can destroy the active conformation of the polymerase. In the following, substances of this kind will be referred to as trans-inhibitors. Such trans-inhibitors include, e.g., heavy metal ions (e.g. Ni, Fe, Mn, Zn etc.), negatively charged polymers (e.g. heparin, dextran sulfate etc.) or protein-denaturing substances which are frequently used in nucleic acid preparations (e.g. SDS, phenol etc.).
However, the sample can also contain substances that bind to nucleic acids thereby inhibiting the amplification, e.g. by preventing denaturation of the nucleic acid or by preventing that the nucleic acid is recognised by a polymerase. In the following, substances of this kind will be referred to as cis-inhibitors. Such cis-inhibitors can be proteins (e.g. histones etc.), positively charged agents (e.g. positively charged polymers, positively charged amino acid etc.).
The nucleic acid can be display various defects so that, at the defect sites, an elongation reaction by the polymerase is no longer possible. These defect sites can be, e.g., single strand or double strand nicks or abasic sites or sites at which modified bases are present (e.g. modification to oxoguanin or uracil in DNA).
At present, the detection of these quantitative and qualitative properties is only possible to an insufficient degree at the beginning of a reaction for the amplification of complex template nucleic acids. It is often not clear in which quality or amount of the complex nucleic acid to be amplified is present. Thus, with very small amounts of complex template nucleic acid, the measurement of concentration cannot be carried out by OD260 measuring. DNA damages can only be determined by gel electrophoresis if the DNA amount is so ample that gel electrophoresis can be carried out. At present it is not possible to determine other damages, such as e.g. abasic DNA segments.
Control of these qualitative parameters of the complex template nucleic acid e.g. via quantitative PCR can be only carried out in an unsatisfactory manner since quantitative PCR allows only poor detection of differences in size for partially degraded DNA if the template nucleic acid has an average size of >1 kb. However, such differences in size can be crucial for the amplification of a complex nucleic acid.